This invention relates to methods for testing the effect of various conditions on oligonucleotide arrays before, during of after production. The invention therefore relates to the fields of quality control in manufacturing and chemical assays.
New technology, called VLSIPS(trademark), has enabled the production of chips smaller than a thumbnail that contain hundreds of thousands or more of different molecular probes. These techniques are described in U.S. Pat. No. 5,143,854, PCT WO 92/10092, and PCT WO 90/15070. Biological chips have probes arranged in arrays, each probe ensemble assigned a specific location. Biological chips have been produced in which each location has a scale of, for example, ten microns. The chips can be used to determine whether target molecules interact with any of the probes on the chip. After exposing the array to target molecules under selected test conditions, scanning devices can examine each location in the array and determine whether a target molecule has interacted with the probe at that location.
Biological chips are useful in a variety of screening techniques for obtaining information about either the probes or the target molecules. For example, a library of peptides can be used as probes to screen for drugs. The peptides can be exposed to a receptor, and those probes that bind to the receptor can be identified.
Biological chips wherein the probes are oligonucleotides (xe2x80x9coligonucleotide arraysxe2x80x9d) show particular promise. Arrays of nucleic acid probes can be used to extract sequence information from nucleic acid samples. The samples are exposed to the probes under conditions that allow hybridization. The arrays are then scanned to determine to which probes the sample molecules have hybridized. One can obtain sequence information by selective tiling of the probes with particular sequences on the arrays, and using algorithms to compare patterns of hybridization and non-hybridization. This method is useful for sequencing nucleic acids. It is also useful in diagnostic screening for genetic diseases or for the presence of a particular pathogen or a strain of pathogen.
The scaled-up manufacturing of oligonucleotide arrays requires application of quality control standards both for determining the quality of chips under current manufacturing conditions and for identifying optimal conditions for their manufacture. Quality control, of course, is not limited to manufacture of chips, but also to the conditions under which they are stored, transported and, ultimately, used.
This invention provides methods for testing the quality of biological chips and the effect of various parameters used in their production by manufacturing oligonucleotide arrays by spatially directed oligonucleotide synthesis in high volume and testing selected arrays. In one embodiment the methods involve determining the extent to which a test condition causes the appearance of a structural feature in oligonucleotides produced on an oligonucleotide array by spatially directed oligonucleotide synthesis by providing a substrate having a surface with linkers having an active site for oligonucleotide synthesis; synthesizing an ensemble of sequence-specific oligonucleotides on the substrate by spatially directed oligonucleotide synthesis, the oligonucleotides optionally having active sites for attaching a detectable label; exposing the area to the test condition; and determining the amount of oligonucleotides having the structural feature.
Certain of the methods of this invention test the efficiency of nucleotide coupling in the synthesis of an oligonucleotide array by spatially directed oligonucleotide synthesis. One of these methods to test efficiency involves providing a substrate having a surface having linkers with active sites; coupling first protected nucleotides to active sites in a first area and second areas of the substrate and capping unreacted, unprotected active sites; deprotecting active sites in the second areas, coupling protected nucleotides to active sites in the second areas and capping unreacted, unprotected active sites in the second areas; optionally repeating the previous step in at least one subsequent area of the substrate and capping unreacted, unprotected active sites in the subsequent areas; determining the amount of competent, uncapped active sites in at least two areas; and comparing the amounts determined. The comparative amount indicates the efficiency of nucleotide coupling between the two areas.
Another method of testing the efficiency of nucleotide coupling involves the steps of providing a substrate having a surface having cleavable linkers including a detectable label and active sites for nucleotide coupling; coupling at least one nucleotide to the active sites and capping unreacted, unprotected active sites after at least one coupling step; cleaving the cleavable linker to release detectably labelled oligonucleotides; determining the lengths of the released oligonucleotides; and comparing the amounts of oligonucleotides having a first length and a second length. The comparative amount indicates the efficiency of nucleotide coupling between the oligonucleotides of the first length and the second length.
This invention also is directed to methods of determining the extent to which a test condition causes deprotection of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis. The method involves the steps of providing a substrate on which an ensemble of sequence-specific oligonucleotides has been synthesized, wherein the active sites on the free terminal nucleotides of the ensemble bear a protecting group; exposing an area of the substrate to the test condition, thereby exposing unprotected active sites from which protective groups have been removed; and determining the amount of unprotected active sites in the area. The amount indicates the extent to which the test condition caused removal of protective groups.
Also provided are methods of determining the extent of depurination of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis. One method involves providing a substrate having a surface with linkers having an active site for oligonucleotide synthesis, the linkers being resistant to cleavage under cleavage conditions; synthesizing an ensemble of sequence-specific oligonucleotides in an area of the substrate, the oligonucleotides having active sites for attaching a detectable label; attaching a detectable label to the oligonuleotides in the ensemble; exposing the ensemble to a test condition; exposing the ensemble to cleavage conditions that cause cleavage of depurinated oligonucleotides; and determining the amount of detectable label in the area.
The other method for testing extent of depurination involves providing a substrate having a surface with linkers having an active site for oligonucleotide synthesis, the linkers being resistant to cleavage under cleavage conditions; synthesizing an ensemble of sequence-specific oligonucleotides in an area of the substrate by spatially directed oligonucleotide synthesis under a test condition, the oligonucleotides having active sites for attaching a detectable label; attaching a detectable label to the active sites; exposing the ensemble to cleavage conditions that cause cleavage of depurinated oligonucleotides; and determining the amount of detectable label in the area.
Another method of this invention is for determining whether an ensemble of oligonucleotides synthesized on a substrate by spatially directed oligonucleotide synthesis contains double-stranded nucleic acids. The method involves providing a substrate on which an ensemble of sequence-specific oligonucleotides has been synthesized in an area of the substrate, the oligonucleotides bearing a detectable label that is released upon cleavage of the oligonucleotide; contacting the ensemble with an agent that cleaves double-stranded nucleic acids, thereby releasing from the substrate detectable label attached to cleaved, double-stranded nucleic acids; and determining the amount of detectable label in the area. The amount of detectable label is inversely related to the amount of double-stranded nucleic acids.
In one embodiment of these methods, spatially directed nucleotide coupling is performed by light-directed nucleotide coupling. In the methods of this invention the step of detecting the amount of certain oligonucleotides on a substrate can involve attaching a detectable label to oligonucleotides in the array. The label can be a fluorescent label, a chemi-luminescent label, a bio-luminescent label, a colorimetric label or a light-scattering label.